Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

Lili Qian,Hegang Li,Dezun Ma,Maoxue Tang,Xiaorong An,Pengfei Gao,Jinzeng Yang,Wentao Cui,Chunbo Cai,Shengwang Jiang,Kui Li,Ke Jiang,Yaoxing Chen,Qingqing Wang

doi:10.1038/srep14435

Abstract

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN−/−) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Highlights

Myostatin (MSTN), known as growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β superfamily, containing three exons and two introns[1,2]
Amino acid sequences of MSTN are highly conserved across species, and the DM phenotype caused by MSTN naturally occurring mutations has been observed in beef cattle such as Belgian Blue and Piedmontese[1,2], sheep[25], dog[26], and human[27], there has been no reports on natural mutations or engineered MSTN mutations with loss of MSTN function and dramatic muscular phenotypes in pigs
Zinc finger nucleases (ZFN) technology overcomes the limitations of embryonic stem cell technology, and allows us to modify the genome of domestic animals with precision and high efficiency[31,32] in combination with somatic cell nucleus transfer (SCNT)

Summary

Introduction

Myostatin (MSTN), known as growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β superfamily, containing three exons and two introns[1,2]. Genetic manipulations of myostatin gene or the use of natural MSTN mutations for livestock meat production have great potentials to increase feed efficiency and healthy food supplies[16]. Amino acid sequences of MSTN are highly conserved across species, and the DM phenotype caused by MSTN naturally occurring mutations has been observed in beef cattle such as Belgian Blue and Piedmontese[1,2], sheep[25], dog[26], and human[27], there has been no reports on natural mutations or engineered MSTN mutations with loss of MSTN function and dramatic muscular phenotypes in pigs. Meishan (Ms) pigs are a locally famous breed in China, and are well known for their high prolificacy and early sexual maturity, but the breed has a high percentage of carcass fat and poor feed efficiency[30] These unique qualities make Ms pig a suitable model to test the effects of MSTN mutations on muscle growth and body composition. Zinc finger nucleases (ZFN) technology overcomes the limitations of embryonic stem cell technology, and allows us to modify the genome of domestic animals with precision and high efficiency[31,32] in combination with somatic cell nucleus transfer (SCNT)

Methods

Results

Conclusion